Fleming was born in Dundee. Her father died when she was young. She married James Orr Fleming and emigrated to America with him. The couple separated and Williamina found herself alone in America with a baby son. She became housekeeper to Edward Pickering, Director of Harvard College Observatory, and then became a member of the Observatory staff. She was the first woman to hold a formal appointment at Harvard.
During her career she classified 10,351 stars, and discovered 10 novae, 52 nebulae and 222 variable stars. In 1906 she was made an honorary member of the Royal Astronomical Society of London.
She died in Boston in 1911.